Purpose: Metastatic melanoma is largely unresponsive to DNA-damaging chemotherapy agents, although WTp53 is frequently detected. Several isoforms of p53 have been discovered, some of which inhibit p53 function. We therefore examined whether p53 isoforms were present in melanoma and whether they may contribute to aberrant p53 function in melanoma. Experimental Design: We studied the expression and subcellular localization of p53 and its isoforms in a panel of human melanoma cell lines using Western blot, two-dimensional electrophoresis, and reverse transcription-PCR. We also characterized the relationship between the expression of p53, p53 isoforms, and p53 target genes following treatment with the DNA-damaging agent cisplatin. Results: We report that p53β and Δ40p53 were expressed in the majority of melanoma cell lines at the mRNA level, but were absent or expressed at low levels in fibroblasts and melanocytes, suggesting that their expression may play a role in melanoma development. Analysis by two-dimensional gel electrophoresis revealed that p53β was expressed at the protein level in melanoma cells. Both p53 and the small molecular weight forms of p53 were aberrantly expressed between the nuclear and cytosolic fractions of melanoma cell lines, compared with normal fibroblasts. Treatment with cisplatin had differential effects on WTp53 and the small molecular weight form of p53 that were cell line dependent. Δ40p53 was shown to inhibit, whereas p53β was shown to enhance, p53-dependent transcription of p21 and PUMA. Conclusions: p53β and Δ40p53 are expressed in melanoma and this may have important implications for understanding resistance of melanoma to DNA-damaging chemotherapy.